- Title
- Contributions of double-stranded RNA binding 2 to the Arabidopsis sRNA landscape
- Creator
- Chamberlain, Kate
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2017
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Small RNA (sRNA)-directed RNA silencing is an essential, conserved mechanism of regulating gene expression in multicellular organisms. Arabidopsis thaliana (Arabidopsis) encodes five DOUBLE-STRANDED RNA BINDING (DRB) proteins, DRB1 to DRB5, which are collectively required for the establishment and maintenance of the sRNA landscape. In the nucleus, DRB1 is the established cofactor of DCL1 for microRNA (miRNA) production from imperfectly double-stranded RNA (dsRNA) precursor transcripts. Similarly, DRB4 is required by DCL4 for the production of species of small-interfering RNA (siRNA) from endogenous or exogenous derived perfectly dsRNA precursors. Despite considerable investigation into the role of DRB2 in RNA silencing, the precise function of this DRB family member remains ambiguous; DRB2 has been implicated in multiple aspects of RNA silencing, including miRNA biogenesis, translation inhibition of miRNA target genes, and the regulation of 24-nt siRNA production. Therefore, the overarching aim of this Thesis was to further characterise the biological function of DRB2 in the parallel sRNA-directed RNA silencing pathways of Arabidopsis. More specifically, the contribution of DRB1, DRB2, and DRB4 to sRNA production, and thereby transcriptional and post-transcriptional RNA silencing, was compared in three experiments. Firstly, the effect of drb mutation on phosphate (PO₄) homeostasis was assessed. The PO4 homeostasis pathway was chosen for analysis as the role of miRNAs in mediating phosphate response has been extensively characterised, which provided an opportunity to compare the effects of DRB1, DRB2, and DRB4 on production of these phosphate responsive miRNAs. In plants, miRNA-directed RNA silencing is central in mediating the response to low inorganic phosphate (Pi) availability, and therefore, it was hypothesised that loss of either DRB1, DRB2 or DRB4 would influence the abundance of PO₄-responsive miRNAs, or the post-transcriptional expression regulation of PO₄-response genes. It was discovered that DRB1 and DRB2 are integral to PO₄ homeostasis in Arabidopsis. However, unlike DRB1, DRB2 does not participate in the production of PO₄-responsive miRNAs. Secondly, the potential association between DRB2 and ARGONAUTE7 (AGO7) in the floral TAS3-derived trans-acting siRNA (TAS3-tasiRNA) pathway was assessed. The drb2ago7 double mutant plant line, generated in this study for the first time, displayed severe reproductive defects, including increased stigma exsertion, reduced seed set, and shortened siliques. These phenotypic alterations strongly indicated a role for DRB2 in the TAS3-tasiRNA pathway. However, it was determined that DRB2 does not participate in the production of the TAS3-tasiRNA ‘trigger’ miRNA, miR390, nor does the drb2 mutation result in altered accumulation of the tasiARF tasiRNA. Rather, the severe defects expressed by the drb2ago7 double mutant are most likely a consequence of altered auxin levels and auxin response gene expression, independent of the TAS3-tasiRNA pathway. Thirdly, sRNA sequencing (sRNA-seq) of floral tissue from single and double drb mutant plants was performed to determine to contribution of each of the five members of the Arabidopsis DRB family on the formation of the Arabidopsis sRNA landscape. Comprehensive bioinformatic analyses revealed the involvement of specific DRB proteins in the production of each of the four major sRNA classes that accumulate in Arabidopsis, including; miRNAs, tasiRNAs, natural antisense siRNAs (natsiRNAs), and PolIV-associated siRNAs (p4-siRNAs). Contrary to the current hypothesis that DRB2 aids DCL1 in miRNA production, it was discovered that DRB2 is not directly involved in miRNA production in Arabidopsis floral tissue. Similarly, DRB2 does not appear to be involved in the production of the tasiRNA or natsiRNA classes. However, loss of DRB2 activity dramatically alters the global abundance of the p4-siRNA class. In drb2 mutant floral tissue, p4-siRNA accumulation is more than two-fold higher across the entire Arabidopsis genome, including the promoter regions and protein-coding sequences of gene bodies, implicating DRB2 in the global regulation of transcriptional RNA silencing. Furthermore, p4-siRNA accumulation was elevated to an even greater degree (3-fold) in transposon-enriched centromeric regions. These findings indicate that DRB2 is antagonistic to p4-siRNA production, as has previously been described. Taken together, the results presented in this Thesis highlight the complexities of the parallel RNA silencing pathways in Arabidopsis, and provide clarification of the role of DRB2 in sRNA production. Specifically, this Thesis presents data that strongly indicates that, in contrast to previous reports, DRB2 does not play a direct role in the production of miRNAs. Instead, the results presented here provide compelling evidence that the primary biological function of DRB2 is to aid in the regulation of the Arabidopsis epigenome.
- Subject
- double-stranded RNA binding; Arabidopsis; small RNA
- Identifier
- http://hdl.handle.net/1959.13/1333582
- Identifier
- uon:27105
- Rights
- Copyright 2017 Kate Chamberlain
- Language
- eng
- Full Text
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| View Details Download | ATTACHMENT01 | Thesis | 8 MB | Adobe Acrobat PDF | View Details Download | ||
| View Details Download | ATTACHMENT02 | Abstract | 358 KB | Adobe Acrobat PDF | View Details Download |